Vulnerability of macaque cranial nerve neurons to ethanol is time- and site-dependent

Abstract

The present study tested the hypotheses that vulnerability to ethanol depends upon (1) population-based characteristics of the neuronal progenitors and (2) the maturation of that population by examining the effects of prenatal exposure to ethanol on brainstem nuclei derived from different rhombomeres and from the alar and basal plates. Macaca nemestrina received an ethanol-containing solution 1 day per week during the first 6 (Et6) or 24 (Et24) weeks of gestation. Control animals received an equivalent volume of saline. The treatment regime for some animals included early gastrulation (gestational day [G] 19 or G20), whereas others were treated later (on G21 or G24). Brainstems were cryosectioned and stained with cresyl violet. Stereological methods were used to determine the numbers of neurons in six different nuclei: the abducens, vagal, and hypoglossal motor nuclei and sensory components of the trigeminal brainstem nuclear complex (the principal, oral, and interpolar subnuclei). There were no differences in the numbers of neurons in any of the nuclei between controls and Et6-, or controls and Et24-treated monkeys. In contrast, the number of trigeminal sensory neurons was significantly (P<.05) lower in animals treated on G19/G20 than in control. No differences between controls and monkeys treated on G21/G24 were detected. No motor nuclei exhibited an ethanol-induced change. These data together with data on the trigeminal motor nucleus show that vulnerability to ethanol (1) is greater in sensory nuclei than in motor nuclei and (2) is temporally restricted to the time of gastrulation.

Figure 1. Schematic diagram of developing brainstem

During development the brainstem transiently forms at least eight rhomobomeres (r1-r8). Sensory neurons are derived from the alar, or roof, plate (right). Motor neurons are derived from the basal, or floor, plate (left).

Figure 2. Appearance of the macaque brainstem

All six nuclei were readily identifiable in horizontal sections stained with cresyl violet. Three sensory nuclei of the trigeminal system are apparent within the same section; the principal sensory nucleus (PSN), and two parts of the spinal trigeminal nucleus - the oralis (SpVo) and the interpolaris (SpVi). The motor nuclei are shown separately. MoVI motor nucleus of the abducens nerve. MoX motor nucleus of the vagus. MoXII motor nucleus of the hypoglossal. Scale bar is 500μm for all images.

Figure 3. The effect of ethanol on the number of sensory neurons

Stereological methods were used to determine volume and cell packing density in each of three motor nuclei. These two parameters were multiplied to generate an estimate of the total number of neurons in each nucleus. Data on the left within each graph are organized according to the duration of exposure; animals were given ethanol on day per week for the first six or all 24 weeks of gestation. On the right within each graph, data are organized based on the day of exposure during the critical time of gastrulation; early gastrulation (gestational day (G)19 or G20) vs. late gastrulation (G21 or G24). Left. The number of neurons in the PSN was significantly (p<0.05) lower in animals exposed to ethanol on G19 or G20 than in control animals. There were no significant differences among control animals and those exposed to ethanol for six or 24 weeks of gestation. Middle. The number of neurons in the spinal trigeminal nucleus oralis was significantly lower in animals exposed to ethanol during early gastrulation compared with control-treated animals. This was a time-dependent effect, and was not seen in animals treated on G21 or G24. There was no significant effect of duration of exposure to ethanol. Right. The time-dependent effect on the number of neurons in the spinal trigeminal nucleus interpolaris was also apparent. Exposure to ethanol during early gastrulation significantly reduced the number of neurons in this nucleus * significantly different to control, p<0.05; # significantly different to animals exposed to ethanol on G21 or G24.

Figure 4. Quantification of the effect of ethanol on motor nuclei

Data were generated and organized as per Figure 3. Exposure to ethanol did not significantly affect the number of neurons in MoVI (left), MoX (middle), or MoXII (right).